Methods for treating dental diseases, disorders and injuries

ABSTRACT

Methods for preventing, reversing, ameliorating or treating dental diseases, disorders or injuries are provided. In particular, methods for preventing, reversing, ameliorating or treating dental diseases of the gingival (gums) and bone are provided. Such methods utilize novel compositions including, but not limited to, extraembryonic cytokine-secreting cells (herein referred to as ECS cells), including, but not limited to, Amnion-derived Multipotent Progenitor cells (herein referred to as AMP cells) and conditioned media derived therefrom (herein referred to as Amnion-derived Cellular Cytokine Solution or ACCS), and Physiologic Cytokine Solution (herein referred to as PCS), each alone or in combination with each other and/or other agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) of U.S.Provisional Application No. 61/459,859, filed Dec. 20, 2010, theentirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

The field of the invention is directed to methods for preventing,reversing, ameliorating or treating dental diseases, disorders andinjuries. In particular, the field of the invention is directed topreventing, reversing, ameliorating or treating dental diseases,disorders or injuries of the gingival (gums) and bone. Such methods forpreventing, reversing, ameliorating and treating such dental diseases,disorders and injuries utilize novel compositions includingextraembryonic cytokine-secreting cells (herein referred to as ECScells) and conditioned medium derived therefrom, includingAmnion-derived Multipotent Progenitor (AMP) cells, conditioned mediumderived therefrom (herein referred to as Amnion-derived CellularCytokine Solution or ACCS, pooled ACCS), and/or cell products derivedtherefrom, and Physiologic Cytokine Solution (herein referred to asPCS), each alone and/or in combination with each other and/or with otheragents including active and/or inactive agents.

BACKGROUND OF THE INVENTION

Gingivitis (inflammation of the gums) usually precedes periodontitis(gum disease). In the early stage of gingivitis, bacteria in plaque (asticky, colorless film of bacteria that forms on teeth) causes the gumsto become inflamed, often bleeding. At this stage the teeth are stillfirmly embedded in their sockets and no irreversible bone or othertissue damage has occurred. However, if left untreated, gingivitis canadvance to periodontitis. In periodontitis, the inner layer of the gumand bone pull away from the teeth and form spaces called pockets. Thesepockets collect debris and often become infected. Toxins, which areproduced by the bacteria in plaque, and enzymes produced by the body asit fights the infection, cause the breakdown of the bone and connectivetissue that anchors the teeth. As the disease progresses, the pocketsdeepen and more bone and connective tissue are destroyed. Eventually theteeth are no longer anchored in place and become loose, often leading totooth loss. In fact, periodontitis is the leading cause of tooth loss inadults.

Plaque is the primary cause of gingivitis and periodontitis. However,other factors can contribute to these diseases as well, includinghormonal changes associated with pregnancy, puberty, menstruation, andmenopause, all of which can make gums more sensitive and easier forgingivitis to develop. In addition, many illnesses can affect the gums.Such illnesses include diseases such as cancer or HIV infection, both ofwhich can interfere with the proper functioning of the immune system.Diabetics are at generally at a higher risk of developing infectionsthan non-diabetics, including periodontal disease. Medications can alsoaffect oral health because some can decrease the flow of saliva, whichhas a protective effect on the teeth and gums. Smoking makes it harderfor gum tissue to repair itself And of course poor oral hygiene such asnot brushing and flossing on a daily basis makes it easier forgingivitis to develop. A family history (genetics) of dental disease canbe a contributing factor for the development of gum disease, as well.

Researchers have identified potential links between gum disease andother serious health conditions such as stroke and heart disease.Diabetes is not only a risk factor for gum disease, but gum disease maymake diabetes worse.

Current non-surgical treatments for gum disease include professionaldental cleaning to remove the plaque and tartar, which is plaque thatbuilds up and hardens on the tooth surface, from above and below the gumline. Often a professional dental cleaning is recommended more thantwice-a-year. Scaling and root planning are deep-cleaning, nonsurgicalprocedures done under a local anesthetic, whereby plaque and tartar fromabove and below the gum line are scraped away (scaling) and rough spotson the tooth root are made smooth (planing) Smoothing the rough spotsremoves bacteria and provides a clean surface for the gums to reattachto the teeth.

Surgical treatments for gum disease include flap surgery/pocketreduction surgery. During this procedure the gums are lifted back andthe tarter is removed. In some cases, irregular surfaces of the damagedbone are smoothed to limit areas where disease-causing bacteria canaccumulate. The gums are then placed so that the tissue fits snuglyaround the tooth. This method reduces the size of the pockets betweenthe gum and tooth, thereby decreasing the areas where harmful bacteriacan grow. Bone grafts involve using fragments of the patients own bone,synthetic bone, or donated bone to replace bone destroyed by gumdisease. The grafts serve as a platform for the regrowth of bone, whichrestores stability to the teeth. New technology, called tissueengineering, encourages the body to regenerate bone and tissue at anaccelerated rate. Soft tissue grafts reinforce thin gums or fills inareas where gums have receded. Grafted tissue, most often taken from theroof of the mouth, is sutured in place, adding tissue to the affectedarea. Guided tissue regeneration is performed when the bone supportingthe teeth has been destroyed. This procedure stimulates bone and gumtissue growth. Done in combination with flap surgery, a small piece ofmesh-like fabric is inserted between the bone and gum tissue. This keepsthe gum tissue from growing into the area where the bone should be,allowing the bone and connective tissue to regrow to better support theteeth. Bone surgery smoothes shallow craters in the bone due to moderateand advanced bone loss. Following flap surgery, the bone around thetooth is reshaped to decrease the craters. This makes it harder forbacteria to collect and grow.

Antibiotic therapy can be used either in combination with surgery andother therapies, or alone, to reduce or temporarily eliminate thebacteria associated with gum disease or suppress the destruction of thetooth's attachment to the bone. Chlorhexidine is an antimicrobial usedto control plaque and gingivitis in the mouth or in periodontal pockets.It is available as a mouth rinse or as a gelatin-filled chip that isplaced in the pockets after root planing and releases the medicationslowly over time. Other antibiotics, including doxycycline,tetracycline, and minocycline may also be used to treat gum disease. Inaddition, a nonprescription toothpaste that contains fluoride and anantibiotic to reduce plaque and gingivitis, called triclosan, may berecommended.

It is an object of the instant invention to provide novel treatmentoptions for patients suffering from dental diseases, disorders orinjuries, in particular, periodontal (gum) diseases, using the novelcompositions described herein. It is also an object of the invention toprovide novel treatment options for patients having other dentaldisorders or conditions such as oral ulcers, including viral ulcers,dental implants, bone grafts, factures of the bone, etc.

BRIEF SUMMARY OF THE INVENTION

It is an object of the instant invention to provide novel methods forpreventing, reversing, ameliorating or treating dental diseases,disorders and injuries, in particular, periodontal (gum) diseases. Suchmethods for preventing, reversing, ameliorating and treating such dentaldiseases, disorders and injuries utilize novel compositions includingextraembryonic cytokine-secreting cells (herein referred to as ECScells) and conditioned medium derived therefrom, includingAmnion-derived Multipotent Progenitor (AMP) cells, conditioned mediumderived therefrom (herein referred to as Amnion-derived CellularCytokine Solution or ACCS, pooled ACCS), and/or cell products derivedtherefrom, and Physiologic Cytokine Solution (herein referred to asPCS), each alone and/or in combination with each other and/or with otheragents including active and/or inactive agents.

Accordingly, a first aspect of the invention is a method for preventing,reversing, ameliorating or treating a dental disease, disorder or injuryin a patient in need thereof comprising administering to the patient atherapeutically effective amount of one or more compositions selectedfrom the group consisting of extraembryonic cytokine-secreting (ECS)cells, conditioned medium derived therefrom, cell lysate derivedtherefrom, cell products derived therefrom, and Physiologic CytokineSolution (PCS).

In one embodiment of aspect 1 the dental disease is selected from thegroup consisting of gingivitis and periodontitis.

In another embodiment of aspect 1 the ECS cells are Amnion-derivedMultipotent Progenitor (AMP) cells.

In yet another embodiment of aspect 1 the conditioned medium isAmnion-derived Cellular Cytokine Solution (ACCS) or pooled ACCS. In aspecific embodiment, the ACCS or pooled ACCS is formulated forsustained-release.

In another specific embodiment of aspect 1 the PCS is formulated forsustained-release.

In still another embodiment of aspect 1 the ECS cells, conditionedmedium derived therefrom, cell lysate derived therefrom or cell productsderived therefrom are administered in combination with other agents ortreatment modalities. In a specific embodiment, the other agents areactive agents. In a particular embodiment the active agents are selectedfrom the group consisting of growth factors, cytokines, inhibitors,immunosuppressive agents, steroids, chemokines, antibodies, antibiotics,antifungals, antivirals, mitomycin C, and other cell types. In anotherspecific embodiment the other treatment modalities are selected from thegroup consisting of non-surgical and surgical treatment modalities. In aparticular embodiment the nonsurgical treatment modalities are selectedfrom the group consisting of professional dental cleaning, scaling androot planning In a particular embodiment the surgical treatmentmodalities are selected from the group consisting of flap surgery/pocketreduction surgery, bone grafts, tissue engineering, soft tissue grafts,guided tissue regeneration, and bone surgery.

Other features and advantages of the invention will be apparent from theaccompanying description, examples and the claims. The contents of allreferences, pending patent applications and issued patents, citedthroughout this application are hereby expressly incorporated byreference. In case of conflict, the present specification, includingdefinitions, will control.

Definitions

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state.

As defined herein, a “gene” is the segment of DNA involved in producinga polypeptide chain; it includes regions preceding and following thecoding region, as well as intervening sequences (introns) betweenindividual coding segments (exons).

As used herein, the term “protein marker” means any protein moleculecharacteristic of a cell or cell population. The protein marker may belocated on the plasma membrane of a cell or in some cases may be asecreted protein.

As used herein, “enriched” means to selectively concentrate or toincrease the amount of one or more materials by elimination of theunwanted materials or selection and separation of desirable materialsfrom a mixture (i.e. separate cells with specific cell markers from aheterogeneous cell population in which not all cells in the populationexpress the marker).

As used herein, the term “substantially purified” means a population ofcells substantially homogeneous for a particular marker or combinationof markers. By substantially homogeneous is meant at least 90%, andpreferably 95% homogeneous for a particular marker or combination ofmarkers.

The term “placenta” as used herein means both preterm and term placenta.

As used herein, the term “totipotent cells” shall have the followingmeaning In mammals, totipotent cells have the potential to become anycell type in the adult body; any cell type(s) of the extraembryonicmembranes (e.g., placenta). Totipotent cells are the fertilized egg andapproximately the first 4 cells produced by its cleavage.

As used herein, the term “pluripotent stem cells” shall have thefollowing meaning Pluripotent stem cells are true stem cells with thepotential to make any differentiated cell in the body, but cannotcontribute to making the components of the extraembryonic membraneswhich are derived from the trophoblast. The amnion develops from theepiblast, not the trophoblast. Three types of pluripotent stem cellshave been confirmed to date: Embryonic Stem (ES) Cells (may also betotipotent in primates), Embryonic Germ (EG) Cells, and EmbryonicCarcinoma (EC) Cells. These EC cells can be isolated fromteratocarcinomas, a tumor that occasionally occurs in the gonad of afetus. Unlike the other two, they are usually aneuploid.

As used herein, the term “multipotent stem cells” are true stem cellsbut can only differentiate into a limited number of types. For example,the bone marrow contains multipotent stem cells that give rise to allthe cells of the blood but may not be able to differentiate into othercells types.

As used herein, the term “extraembryonic tissue” means tissue locatedoutside the embryonic body which is involved with the embryo'sprotection, nutrition, waste removal, etc. Extraembryonic tissue isdiscarded at birth. Extraembryonic tissue includes but is not limited tothe amnion, chorion (trophoblast and extraembryonic mesoderm includingumbilical cord and vessels), yolk sac, allantois and amniotic fluid(including all components contained therein). Extraembryonic tissue andcells derived therefrom have the same genotype as the developing embryo.

As used herein, the term “extraembryonic cells” or “EE cells” means apopulation of cells derived from the extraembryonic tissue.

As used herein, the term “extraembryonic cytokine-secreting cells” or“ECS cells” means a population of cells derived from the extraembryonictissue which have the characteristic of secreting VEGF, Angiogenin, PDGFand TGFβ32 and the MMP inhibitors TIMP-1 and/or TIMP-2 atphysiologically relevant levels in a physiologically relevant temporalmanner into the extracellular space or into the surrounding culturemedia. ECS cells have not been cultured in the presence of any non-humananimal materials, making them and cell products derived from themsuitable for human clinical use as they are not xeno-contaminated. ECScells may be selected from populations of cells and compositionsdescribed in this application and in US2003/0235563, US2004/0161419,US2005/0124003, U.S. Provisional Application Nos. 60/666,949,60/699,257, 60/742,067, 60/813,759, U.S. application Ser. No.11/333,849, U.S. application Ser. No. 11/392,892, PCTUS06/011392,US2006/0078993, PCT/US00/40052, U.S. Pat. No. 7,045,148, US2004/0048372,and US2003/0032179, the contents of which are incorporated herein byreference in their entirety. ECS cells were previously called trophicfactor-secreting extraembryonic cells or TSE cells.

As used herein, the term “Amnion-derived Multipotent Progenitor cell” or“AMP cell” means a specific population of cells that are epithelialcells derived from the amnion. AMP cells have the followingcharacteristics. They have not been cultured in the presence of anynon-human animal materials, making them and cell products derived fromthem suitable for human clinical use as they are not xeno-contaminated.AMP cells are cultured in basal medium supplemented with human serumalbumin. In a preferred embodiment, the AMP cells secrete the cytokinesVEGF, Angiogenin, PDGF and TGFβ32 and the MMP inhibitors TIMP-1 and/orTIMP-2. The physiological range of the cytokine or cytokines in theunique combination is as follows: ˜5-16 ng/mL for VEGF, ˜3.5-4.5 ng/mLfor Angiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ32,˜0.68 μg/mL for TIMP-1 and ˜1.04 μg/mL for TIMP-2. The AMP cells mayoptionally express Thymosin 134. AMP cells grow without feeder layers,do not express the protein telomerase and are non-tumorigenic. AMP cellsdo not express the hematopoietic stem cell marker CD34 protein. Theabsence of CD34 positive cells in this population indicates the isolatesare not contaminated with hematopoietic stem cells such as umbilicalcord blood or embryonic fibroblasts. Virtually 100% of the cells reactwith antibodies to low molecular weight cytokeratins, confirming theirepithelial nature. Freshly isolated amnion-derived cells, from which AMPcells are selected and isolated, will not react with antibodies to thestem/progenitor cell markers c-kit (CD117) and Thy-1 (CD90). Severalprocedures used to obtain cells from full term or pre-term placenta areknown in the art (see, for example, US 2004/0110287; Anker et al., 2005,Stem Cells 22:1338-1345; Ramkumar et al., 1995, Am. J. Ob. Gyn.172:493-500). However, the methods used herein provide improved andunique compositions and populations of cells.

By the term “animal-free” when referring to certain compositions, growthconditions, culture media, etc. described herein, is meant that nonon-human animal-derived materials, such as bovine serum, proteins,lipids, carbohydrates, nucleic acids, vitamins, etc., are used in thepreparation, growth, culturing, expansion, storage or formulation of thecertain composition or process. By “no non-human animal-derivedmaterials” is meant that the materials have never been in or in contactwith a non-human animal body or substance so they are notxeno-contaminated. Only clinical grade materials, such as recombinantlyproduced human proteins, are used in the preparation, growth, culturing,expansion, storage and/or formulation of such compositions and/orprocesses.

By the term “expanded”, in reference to cell compositions, means thatthe cell population constitutes a significantly higher concentration ofcells than is obtained using previous methods. For example, the level ofcells per gram of amniotic tissue in expanded compositions of AMP cellsis at least 50 and up to 150 fold higher than the number of amnionepithelial cells in the primary culture after 5 passages, as compared toabout a 20 fold increase in such cells using previous methods. Inanother example, the level of cells per gram of amniotic tissue inexpanded compositions of AMP cells is at least 30 and up to 100 foldhigher than the number of amnion epithelial cells in the primary cultureafter 3 passages. Accordingly, an “expanded” population has at least a 2fold, and up to a 10 fold, improvement in cell numbers per gram ofamniotic tissue over previous methods. The term “expanded” is meant tocover only those situations in which a person has intervened to elevatethe number of the cells.

As used herein, the term “passage” means a cell culture technique inwhich cells growing in culture that have attained confluence or areclose to confluence in a tissue culture vessel are removed from thevessel, diluted with fresh culture media (i.e. diluted 1:5) and placedinto a new tissue culture vessel to allow for their continued growth andviability. For example, cells isolated from the amnion are referred toas primary cells. Such cells are expanded in culture by being grown inthe growth medium described herein. When such primary cells aresubcultured, each round of subculturing is referred to as a passage. Asused herein, “primary culture” means the freshly isolated cellpopulation.

As used herein, the term “differentiation” means the process by whichcells become progressively more specialized.

As used herein, the term “differentiation efficiency” means thepercentage of cells in a population that are differentiating or are ableto differentiate.

As used herein, “conditioned medium” is a medium in which a specificcell or population of cells has been cultured, and then removed. Whencells are cultured in a medium, they may secrete cellular factors thatcan provide support to or affect the behavior of other cells. Suchfactors include, but are not limited to hormones, cytokines,extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines,receptors, inhibitors and granules. The medium containing the cellularfactors is the conditioned medium.

As used herein, the term “Amnion-derived Cellular Cytokine Solution” or“ACCS” means conditioned medium that has been derived from AMP cellsthat have been cultured in basal media supplemented with human serumalbumin. ACCS has previously been referred to as “amnion-derivedcellular cytokine suspension”.

The term “physiological level” as used herein means the level that asubstance in a living system is found and that is relevant to the properfunctioning of a biochemical and/or biological process.

As used herein, the term “Physiologic Cytokine Solution” or “PCS”composition means a composition which is not cell-derived and which hasphysiologic concentrations of one or more factors selected from VEGF,Angiogenin, PDGF and TGFβ32 and at least one MMP inhibitor. Examples ofsuitable MMP inhibitors include but are not limited to TIMP-1 andTIMP-2. Details on PCS can be found in U.S. Publication No.US-2009-0054339-A1, the contents of which are incorporated herein byreference.

As used herein, the term “pooled” means a plurality of compositions thathave been combined to create a new composition having more constant orconsistent characteristics as compared to the non-pooled compositions.

The term “therapeutically effective amount” means that amount of atherapeutic agent necessary to achieve a desired physiological effect(i.e. treat dental disease).

The term “lysate” as used herein refers to the composition obtained whencells, for example, AMP cells, are lysed and optionally the cellulardebris (e.g., cellular membranes) is removed. This may be achieved bymechanical means, by freezing and thawing, by sonication, by use ofdetergents, such as EDTA, or by enzymatic digestion using, for example,hyaluronidase, dispase, proteases, and nucleases. In some instances, itmay be desirable to lyse the cells and retain the cellular membraneportion and discard the remaining portion of the lysed cells.

As used herein, the term “pharmaceutically acceptable” means that thecomponents, in addition to the therapeutic agent, comprising theformulation, are suitable for administration to the patient beingtreated in accordance with the present invention.

As used herein, the term “tissue” refers to an aggregation of similarlyspecialized cells united in the performance of a particular function.

As used herein, the term “therapeutic protein” includes a wide range ofbiologically active proteins including, but not limited to, growthfactors, enzymes, hormones, cytokines, inhibitors of cytokines, bloodclotting factors, peptide growth and differentiation factors.

The term “transplantation” as used herein refers to the administrationof a composition comprising cells, including a cell suspension or cellsincorporated into a matrix or tissue, that are either in anundifferentiated, partially differentiated, or fully differentiated forminto a human or other animal.

As used herein, the terms “a” or “an” means one or more; at least one.

As used herein, the term “adjunctive” means jointly, together with, inaddition to, in conjunction with, and the like.

As used herein, the term “co-administer” can include simultaneous orsequential administration of two or more agents.

“Treatment,” “treat,” or “treating,” as used herein covers any treatmentof a disease or condition of a mammal, particularly a human, andincludes: (a) preventing the disease or condition from occurring in asubject which may be predisposed to the disease or condition but has notyet been diagnosed as having it; (b) inhibiting the disease orcondition, i.e., arresting its development; (c) relieving and orameliorating the disease or condition, i.e., causing regression of thedisease or condition; or (d) curing the disease or condition, i.e.,stopping its development or progression. The population of subjectstreated by the methods of the invention includes subjects suffering fromthe undesirable condition or disease, as well as subjects at risk fordevelopment of the condition or disease. The term “ameliorate” as usedherein means to improve, make better, make more tolerable or reverse acondition, for example, a dental disease, disorder or injury.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984,“Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized CellsAnd Enzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Therapeutic Uses—The compositions of the invention are useful inpreventing, reversing, ameliorating or treating dental diseases,disorders, or injuries, including but not limited to gingivitis andperiodontitis.

Obtaining and Culturing of Cells

ECS cells—Various methods for isolating cells from the extraembryonictissue, which may then be used to produce the ECS cells of the instantinvention are described in the art (see, for example, US2003/0235563,US2004/0161419, US2005/0124003, U.S. Provisional Application Nos.60/666,949, 60/699,257, 60/742,067, 60/813,759, U.S. application Ser.No. 11/333,849, U.S. application Ser. No. 11/392,892, PCTUS06/011392,US2006/0078993, PCT/US00/40052, U.S. Pat. No. 7,045,148, US2004/0048372,and US2003/0032179).

Identifying ECS cells—Once extraembryonic tissue is isolated, it isnecessary to identify which cells in the tissue have the characteristicsassociated with ECS cells (see definition above). For example, cells areassayed for their ability to secrete VEGF, Angiogenin, PDGF and TGFβ32and the MMP inhibitors TIMP-1 and/or TIMP-2 into the extracellular spaceor into surrounding culture media. In some instances, it may bedifficult or impossible to detect certain factors using standard assays.This may be because certain factors are secreted by the cells atphysiological levels that are below the level of detection by the assaymethods. It may also be that the factor(s) is being utilized by the ECScell and/or by other local cells, thus preventing accumulation atdetectable levels using standard assays. It is also possible that thetemporal manner in which the factors are secreted may not coincide withthe timing of sampling.

AMP cell compositions are prepared using the steps of a) recovery of theamnion from the placenta, b) dissociation of the epithelial cells fromthe amniotic membrane using a protease, c) culturing of the cells in abasal medium with the addition of a naturally derived or recombinantlyproduced human serum albumin and no non-human animal protein; d)selecting AMP cells from the epithelial cell culture, and optionally e)further proliferation of the cells, optionally using additionaladditives and/or growth factors (i.e. recombinant human EGF). Detailsare contained in US Publication No. 2006-0222634-A1, which isincorporated herein by reference.

Culturing of the AMP cells—The cells are cultured in a basal medium.Such medium includes, but is not limited to, EPILIFE® culture medium forepithelial cells (Cascade Biologicals), OPTI-PRO™ serum-free culturemedium, VP-SFM serum-free medium, IMDM highly enriched basal medium,KNOCKOUT™ DMEM low osmolality medium, 293 SFM II defined serum-freemedium (all made by Gibco; Invitrogen), HPGM hematopoietic progenitorgrowth medium, Pro 293S-CDM serum-free medium, Pro 293A-CDM serum-freemedium, UltraMDCK™ serum-free medium (all made by Cambrex), STEMLINE®T-cell expansion medium and STEMLINE® II hematopoietic stem cellexpansion medium (both made by Sigma-Aldrich), DMEM culture medium,DMEM/F-12 nutrient mixture growth medium (both made by Gibco), Ham'sF-12 nutrient mixture growth medium, M199 basal culture medium (bothmade by Sigma-Aldrich), and other comparable basal media. Such mediashould either contain human protein or be supplemented with humanprotein. As used herein a “human protein” is one that is producednaturally or one that is produced using recombinant technology, forexample, human serum albumin. In specific embodiments, the basal mediais IMDM highly enriched basal medium, STEMLINE® T-cell expansion mediumor STEMLINE® II hematopoietic stem cell expansion medium, or OPTI-PRO™serum-free culture medium, or combinations thereof and the human proteinis human serum albumin added at at least 0.5% and up to 10%. Inparticular embodiments, the human serum albumin is from about 0.5% toabout 2%. In a specific embodiment the human serum albumin is at 0.5%.The human serum albumin may come from a liquid or a dried (powder) formand includes, but is not limited to, recombinant human serum albumin,PLASBUMIN® normal human serum albumin and PLASMANATE® human bloodfraction (both made by Talecris Biotherapeutics).

In a most preferred embodiment, the cells are cultured using a systemthat is free of non-human animal products to avoid xeno-contamination.In this embodiment, the culture medium is IMDM highly enriched basalmedium , STEMLINE® T-cell expansion medium or STEMLINE® II hematopoieticstem cell expansion medium, OPTI-PRO™ serum-free culture medium, or DMEMculture medium, with human serum albumin added up to amounts of 10%.

The invention further contemplates the use of any of the above basalmedia wherein animal-derived proteins are replaced with recombinanthuman proteins and animal-derived serum, such as BSA, is replaced withhuman serum albumin. In preferred embodiments, the media is serum-freein addition to being non-human animal-free.

Optionally, other factors are used. In one embodiment, epidermal growthfactor (EGF) at a concentration of between 0-1 μg/mL is used. In apreferred embodiment, the EGF concentration is around 10-20 ng/mL.

Generation of Conditioned Medium

ECS cell conditioned medium—is obtained as described below for ACCS,except that ECS cells are used.

Generation of ACCS—The AMP cells of the invention can be used togenerate ACCS. In one embodiment, the AMP cells are isolated asdescribed herein and 1×10⁶ cells/mL are seeded into T75 flaskscontaining between 5-30 mL culture medium, preferably between 10-25 mLculture medium, and most preferably about 10 mL culture medium. Thecells are cultured until confluent, the medium is changed and in oneembodiment the ACCS is collected 1 day post-confluence. In anotherembodiment the medium is changed and ACCS is collected 2 dayspost-confluence. In another embodiment the medium is changed and ACCS iscollected 3 days post-confluence. In another embodiment the medium ischanged and ACCS is collected 4 days post-confluence. In anotherembodiment the medium is changed and ACCS is collected 5 dayspost-confluence. In another embodiment the medium is changed and ACCS iscollected 3 days post-confluence. In another preferred embodiment themedium is changed and ACCS is collected 3, 4, 5, 6 or more dayspost-confluence. Skilled artisans will recognize that other embodimentsfor collecting ACCS from AMP cell cultures, such as using other tissueculture vessels, including but not limited to cell factories, flasks,hollow fibers, or suspension culture apparatus, or collecting ACCS fromsub-confluent and/or actively proliferating cultures, are alsocontemplated by the methods of the invention. It is also contemplated bythe instant invention that the ACCS be cryopreserved followingcollection. It is also contemplated by the invention that ACCS belyophilized following collection. It is also contemplated that ACCS beformulated for sustained-release after collection.

The compositions of the invention can be prepared in a variety of waysdepending on the intended use of the compositions. For example, acomposition useful in practicing the invention may be a liquidcomprising an agent of the invention, i.e. ECS cells, including AMPcells and/or ACCS, pooled ACCS or PCS, in solution, in suspension, orboth (solution/suspension). The term “solution/suspension” refers to aliquid composition where a first portion of the active agent is presentin solution and a second portion of the active agent is present inparticulate form, in suspension in a liquid matrix. A liquid compositionalso includes a gel. The liquid composition may be aqueous or in theform of an ointment, salve, cream, or the like.

An aqueous suspension or solution/suspension useful for practicing themethods of the invention may contain one or more polymers as suspendingagents. Useful polymers include water-soluble polymers such ascellulosic polymers and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. An aqueous suspension orsolution/suspension of the present invention is preferably viscous ormuco-adhesive, or even more preferably, both viscous and muco-adhesive.

Pharmaceutical Compositions—The present invention providespharmaceutical compositions of ECS cells, including AMP cells and/orACCS, pooled ACCS or PCS and a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly, in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the composition isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, and still othersare familiar to skilled artisans.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Treatment Kits—The invention also provides for an article of manufacturecomprising packaging material and a pharmaceutical composition of theinvention contained within the packaging material, wherein thepharmaceutical composition comprises compositions of ECS cells,including AMP cells and/or ACCS, pooled ACCS or PCS. The packagingmaterial comprises a label or package insert which indicates that theECS cells, including AMP cells and/or ACCS, pooled ACCS or PCS can beused for preventing, reversing, ameliorating or treating dentaldiseases, disorders or injuries.

Formulation, Dosage and Administration

Compositions comprising ECS cells, including AMP cells and/or ACCS,pooled ACCS or PCS may be administered to a subject to provide variouscellular or tissue functions, for example, to prevent, reverse,ameliorate or treat dental diseases, disorders or injuries. As usedherein “subject” may mean either a human or non-human animal.

Such compositions may be formulated in any conventional manner using oneor more physiologically acceptable carriers optionally comprisingexcipients and auxiliaries. Proper formulation is dependent upon theroute of administration chosen. The compositions may be packaged withwritten instructions for their use in preventing, reversing or treatingdental diseases or restoring a therapeutically important metabolicfunction. The compositions may also be administered to the recipient inone or more physiologically acceptable carriers. Carriers for the cellsmay include but are not limited to solutions of phosphate bufferedsaline (PBS) or lactated Ringer's solution containing a mixture of saltsin physiologic concentrations.

Pharmaceutical compositions useful in the practice of certainembodiments of the invention (i.e. those utilizing topicaladministration) include a therapeutically effective amount of an activeagent with a pharmaceutically acceptable carrier. Such pharmaceuticalcompositions may be liquid, gel, ointment, salve, sustained-releaseformulations or other formulations suitable for administration to thegums, bone and teeth. The composition comprises a composition of theinvention (i.e. ECS cells, including AMP cells and/or ACCS, pooled ACCSor PCS) and, optionally, at least one pharmaceutically acceptableexcipient.

In various embodiments, compositions of the invention can comprise aliquid comprising an active agent in solution, in suspension, or both.The term “suspension” herein includes a liquid composition wherein afirst portion of the active agent is present in solution and a secondportion of the active agent is present in particulate form, insuspension in a liquid matrix. As used herein, liquid compositionsinclude gels.

Preferably the liquid composition is aqueous. Alternatively, thecomposition can take form of an ointment. In a preferred embodiment, thecomposition is an in situ gellable aqueous composition, more preferablyan in situ gellable aqueous solution. Such a composition can comprise agelling agent in a concentration effective to promote gelling uponcontact with the gums and/or teeth. Suitable gelling agentsnon-restrictively include thermosetting polymers such astetra-substituted ethylene diamine block copolymers of ethylene oxideand propylene oxide (e.g., poloxamine 1307); polycarbophil; andpolysaccharides such as gellan, carrageenan (e.g., kappa-carrageenan andiota-carrageenan), chitosan and alginate gums. The phrase “in situgellable” includes not only liquids of low viscosity that can form gels,but also more viscous liquids such as semi-fluid and thixotropic gelsthat exhibit substantially increased viscosity or gel stiffness uponadministration.

Aqueous compositions of the invention have physiologically compatible pHand osmolality. Preferably these compositions incorporate means toinhibit microbial growth, for example through preparation and packagingunder sterile conditions and/or through inclusion of an antimicrobiallyeffective amount of an acceptable preservative. Suitable preservativesnon-restrictively include mercury-containing substances such asphenylmercuric salts (e.g., phenylmercuric acetate, borate and nitrate)and thimerosal; stabilized chlorine dioxide; quaternary ammoniumcompounds such as benzalkonium chloride, cetyltrimethylammonium bromideand cetylpyridinium chloride; imidazolidinyl urea; parabens such asmethylparaben, ethylparaben, propylparaben and butylparaben, and saltsthereof; phenoxyethanol; chlorophenoxyethanol; phenoxypropanol;chlorobutanol; chlorocresol; phenylethyl alcohol; disodium EDTA; andsorbic acid and salts thereof.

The composition can comprise a depot formulation comprising an activeagent for topical administration. The depot formulation comprises acomposition of the invention (i.e. ECS cells, including AMP cells and/orACCS, pooled ACCS or PCS). The microparticles comprising thecompositions can be embedded in a biocompatible pharmaceuticallyacceptable polymer or a lipid encapsulating agent. The depotformulations may be adapted to release all of substantially all theactive material over an extended period of time. The polymer or lipidmatrix, if present, may be adapted to degrade sufficiently to betransported from the site of administration after release of all orsubstantially all the active agent. The depot formulation can be liquidformulation, comprising a pharmaceutical acceptable polymer and adissolved or dispersed active agent. Upon injection, the polymer forms adepot at the injections site, e.g. by gelifying or precipitating.

The composition can comprise a solid article that can be inserted in asuitable location in the mouth, where the article releases the activeagent. Release from such an article is preferably to the teeth, gums andbone, with which the solid article is generally in intimate contact.Solid articles suitable for implantation in the mouth in such fashiongenerally comprise polymers and can be bioerodible or non-bioerodible.Bioerodible polymers that can be used in preparation of implantscarrying a composition in accordance with the present invention includewithout restriction aliphatic polyesters such as polymers and copolymersof poly(glycolide), poly(lactide), poly(.epsilon.-caprolactone),poly(hydroxybutyrate) and poly(hydroxyvalerate), polyamino acids,polyorthoesters, polyanhydrides, aliphatic polycarbonates and polyetherlactose. Illustrative of suitable non-bioerodible polymers are siliconeelastomers.

One of skill in the art may readily determine the appropriateconcentration, or dose, of the ECS cells, including AMP cells and/orACCS, pooled ACCS or PCS, for a particular purpose. The skilled artisanwill recognize that a preferred dose is one which produces a therapeuticeffect, such as preventing, reversing, ameliorating or treating dentaldiseases, disorders or injuries in a patient in need thereof. Forexample, one preferred dose of ACCS, pooled ACCS or PCS is in the rangeof about 0.1-to-1000 μL per square centimeter of applied area. Otherpreferred dose ranges are 1.0-100 μL per square centimeter of appliedarea and about 0.01-to-50.0 μL per square centimeter of applied area.Likewise, ECS cells, including AMP cells, are prepared at aconcentration of between about 1×10⁷-1×10⁸ cells/mL, preferably at about2.5×10⁷-7.5×10⁷ cells/mL, and most preferably at about 5×10⁷ cells/mL.Of course, proper doses of the ECS cells, including AMP cells and/orACCS, pooled ACCS or PCS, will require empirical determination at timeof use based on several variables including but not limited to theseverity and type of disease, injury, disorder or condition beingtreated; patient age, weight, sex, health; other medications andtreatments being administered to the patient; and the like. One of skillin the art will also recognize that number of doses (dosing regimen) tobe administered needs also to be empirically determined based on, forexample, severity and type of disease, injury, disorder or conditionbeing treated. In a preferred embodiment, one dose is sufficient. Otherpreferred embodiments contemplate, 2, 3, 4, or more doses.

In further embodiments of the present invention, it may be desirable toco-administer other agents, including active agents and/or inactiveagents, with the ECS cells, including AMP cells and/or ACCS, pooled ACCSor PCS, to prevent, reverse, ameliorate or treat dental diseases. Activeagents include but are not limited to cytokines, chemokines, antibodies,inhibitors, antibiotics, anti-fungals, anti-virals, immunosuppressiveagents, other cell types, and the like. Inactive agents includecarriers, diluents, stabilizers, gelling agents, delivery vehicles, ECMs(natural and synthetic), scaffolds, and the like. When the ECS cells,including AMP cells and/or ACCS, pooled ACCS or PCS, are administeredconjointly with other pharmaceutically active agents, even less of theECS cells, including AMP cells and/or ACCS, pooled ACCS or PCS, may beneeded to be therapeutically effective.

ECS cells, including AMP cells and/or ACCS, pooled ACCS or PCS, can beadministered by injection into a target site of a subject, preferablyvia a delivery device, such as a tube, e.g., catheter. In a preferredembodiment, the tube additionally contains a needle, e.g., a syringe,through which the cells and/or ACCS or PCS can be introduced into thesubject at a desired location.

The timing of administration of ECS cells, including AMP cells and/orACCS, pooled ACCS or PCS, will depend upon the type and severity of thedental disease being treated. In a preferred embodiment, the ECS cells,including AMP cells and/or ACCS, pooled ACCS or PCS, are administered assoon as possible after the dental disease is diagnosed. In otherpreferred embodiments, the ECS cells, including AMP cells and/or ACCS,pooled ACCS or PCS, are administered more than one time followingdiagnosis.

Also contemplated by the methods of the invention are compositionscomprising cells that have been partially or fully differentiated. Suchpartially or fully differentiated cell compositions are obtained bytreating ECS cells, including AMP cells, with appropriate reagents andunder appropriate conditions wherein the cells undergo partial orcomplete differentiation into, for example, connective tissue cells suchas, for example, fibroblasts or bone cells. Skilled artisans arefamiliar with conditions capable of effecting such partial or completedifferentiation. The cells may be treated under differentiatingconditions prior to use (i.e. prior to transplantation, administration,etc.) or simultaneously with use. In certain embodiments, the cells aretreated under differentiation conditions before and during use.

Sustained-Release Compositions

The ACCS, pooled ACCS or PCS, maybe formulated as sustained-releasecompositions. Skilled artisans are familiar with methodologies to createsustained-release compositions of therapeutic agents, includingprotein-based therapeutic agents such as ACCS, pooled ACCS or PCS.

The sustained-release compositions may be made by any of the methodsdescribed herein. For example, multivesicular liposome formulationtechnology is useful for the sustained-release of protein and peptidetherapeutics. Qui, J., et al, (ACTA Pharmacol Sin, 2005,26(11):1395-401) describe this methodology for the formulation ofsustained-release interferon alpha-2b. Vyas, S. P., et al, (Drug Dev IndPharm, 2006, 32(6):699-707) describe encapsulating pegylated interferonalpha in multivesicular liposomes. ACCS, including pooled ACCS, and PCSare suitable for use in multivesicular liposome sustained-releaseformulation.

Nanoparticle technology is also useful for creating sustained-releasecompositions. For example, Packhaeuser, C. B., et al, (J ControlRelease, 2007, 123(2):131-40) describe biodegradable parenteral depotsystems based on insulin loaded dialkylaminoalkyl-amine-poly(vinylalcohol)-g-poly(lactide-co-glycolide) nanoparticules and conclude thatnanoparticle-based depots are suitable candidates for the design ofcontrolled-release devices for bioactive macromolecules (i.e. proteins).Dailey, L. A., et al, (Pharm Res 2003, 20(12):2011-20) describesurfactant-free, biodegradable nanoparticles for aerosol therapy whichis based on the branched polymers DEAPA-PVAL-g-PLGA and conclude thatDEAPA-PVAL-g-PLGA are versatile drug delivery systems. ACCS, includingpooled ACCS, and PCS are suitable for use in nanoparticle-basedsustained-release formulations.

Polymer-based sustained-release formulations are also very useful. Chan,Y. P., et al, (Expert Opin Drug Deliv, 2007, 4(4):441-51) provide areview of the Medusa system (Flamel Technologies), which is used forsustained-release of protein and peptide therapies. Thus far, the Medusasystem has been applied to subcutaneous injection of IL-2 andIFN-alpha(2b), in animal models (rats, dogs, monkeys), and in clinicaltrials in renal cancer (IL-2) and hepatitis C (IFN-alpha(2b)) patients.Chavanpatil, M. D., et al, (Pharm Res, 2007, 24(4):803-10) describesurfactant-polymer nanoparticles as a novel platform for sustained andenhanced cellular delivery of water-soluble molecules. Takeuchi, H., etal, (Adv Drug Deliv Res, 2001, 47(1):39-54) describe mucoadhesivenanoparticulate systems for peptide drug delivery, including liposomesand polymeric nanoparticles. Wong, H. L., et al, (Pharm Res, 2006,23(7):1574-85) describe a new polymer-lipid hybrid system which has beenshown to increase cytotoxicity of doxorubicin againstmultidrug-resistant breast cancer cells. ACCS, including pooled ACCS,and PCS are suitable for use in the aforementioned sustained-releaseformulation methodologies.

In addition, other sustained-release methodologies familiar to skilledartisans, while not specifically described herein, are also suitable foruse.

Skilled artisans will recognize that any and all of the standard methodsand modalities for preventing, reversing or treating dental diseasescurrently in clinical practice and clinical development are suitable forpracticing the methods of the invention. Routes of administration,formulation, co-administration with other agents (if appropriate) andthe like are discussed in detail elsewhere herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Preparation of AMP Cell Compositions

Amnion epithelial cells were dissociated from starting amniotic membraneusing the dissociation agents PXXIII. The average weight range of anamnion was 18-27 g. The number of cells recovered per g of amnion wasabout 10-15×10⁶ for dissociation with PXXIII.

Method of obtaining selected AMP cells—Amnion epithelial cells wereplated immediately upon isolation from the amnion. After ˜2 days inculture non-adherent cells were removed and the adherent cells werekept. This attachment to a plastic tissue culture vessel is theselection method used to obtain the desired population of AMP cells.Adherent and non-adherent AMP cells appear to have a similar cellsurface marker expression profile but the adherent cells have greaterviability and are the desired population of cells. Adherent AMP cellswere cultured in basal medium supplemented with human serum albuminuntil they reached ˜120,000-150,000 cells/cm². At this point, thecultures were confluent. Suitable cell cultures will reach this numberof cells between ˜5-14 days. Attaining this criterion is an indicator ofthe proliferative potential of the AMP cells and cells that do notachieve this criterion are not selected for further analysis and use.Once the AMP cells reached ˜120,000-150,000 cells/cm², they werecollected and cryopreserved. This collection time point is called p0.

Example 2 Generation of ACCS

The AMP cells of the invention can be used to generate ACCS, includingpooled ACCS. The AMP cells were isolated as described above and ˜1×10⁶cells/mL were seeded into T75 flasks containing ˜10 mL culture medium asdescribed above. The cells were cultured until confluent, the medium waschanged and ACCS was collected 3 days post-confluence. Optionally, theACCS is collected again after 3 days, and optionally again after 3 days.Skilled artisans will recognize that other embodiments for collectingACCS from confluent cultures, such as using other tissue culturevessels, including but not limited to cell factories, flasks, hollowfibers, or suspension culture apparatus, etc. are also contemplated bythe methods of the invention (see Detailed Description above). It isalso contemplated by the instant invention that the ACCS becryopreserved, lyophilized, irradiated and/or formulated forsustained-release following collection. It is also contemplated thatACCS be collected at different time points (see Detailed Description fordetails).

Example 3 Generation of PCS Compositions

The following PCS compositions are produced by combining the indicatedcytokine or factor at physiologic levels in a carrier:

Composition A: VEGF and TIMP-1; Composition B: VEGF, Angiogenin andTIMP-1; Composition C: VEGF, Angiogenin, PDGF-BB and TIMP-1; CompositionD: VEGF, Angiogenin, PDGF-BB, TGFβ32 and TIMP-1; Composition E: VEGF andTIMP-2; Composition F: VEGF, Angiogenin and TIMP-2; Composition G: VEGF,Angiogenin, PDGF-BB and TIMP-2; Composition H: VEGF, Angiogenin,PDGF-BB, TGFβ2 and TIMP-2; Composition I: VEGF, TIMP-1 and TIMP-2;Composition J: VEGF, Angiogenin, TIMP-1 and TIMP-2; Composition K: VEGF,Angiogenin, PDGF-BB, TIMP-1 and TIMP-2; Composition L: VEGF, Angiogenin,PDGF-BB, TGFβ2, TIMP-1 and TIMP-2; Composition M: Angiogenin and TIMP-1;Composition N: Angiogenin, PDGF-BB and TIMP-1; Composition 0:Angiogenin, PDGF-BB, TGFβ2 and TIMP-1; Composition P: Angiogenin andTIMP-2; Composition Q: Angiogenin, PDGF-BB and TIMP-2; Composition R:Angiogenin, PDGF-BB, TGFβ2 and TIMP-2; Composition S: Angiogenin,PDGF-BB, TGFβ2, TIMP-1 and TIMP-2; Composition T: PDGF-BB and TIMP-1;Composition U: PDGF-BB, TGFβ2 and TIMP-1; Composition V: PDGF-BB andTIMP-2; Composition W: PDGF-BB, TGFβ2 and TIMP-2; Composition X:PDGF-BB, TIMP-1 and TIMP-2; Composition Y: PDGF-BB, TGFβ2, TIMP-1 andTIMP-2. A preferred composition is Composition L.

Compositions A-Y optionally contains Thymosin P4. Skilled artisans willrecognize that in certain embodiments other MMP inhibitors (i.e. TIMP-3,TIMP-4 or synthetic MMP inhibitors) may be suitable (J. FrederickWoessner, Jr., J. Clin. Invest. 108(6): 799-800 (2001); Brew, K., et al,Biochim Biophys Acta. 2000 Mar 7;1477(1-2):267-83).

VEGF, Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2 are added at thefollowing physiologic levels: ˜5-16 ng/mL for VEGF, ˜3.5-4.5 ng/mL forAngiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68 μgmL for TIMP-1 and ˜1.04 μg/mL for TIMP-2. VEGF may be obtained fromInvitrogen, catalog # PHG0144, PHG0145, PHG0146, PHG0141 or PHG0143;Angiogenin may be obtained from R&D Systems, catalog #265-AN-050 or265-AN-250; PDGF-BB may be obtained from Invitrogen, catalog # PHG0044,# PHG0045, # PHG0046, # PHG0041, # PHG0043; TGFβ2 may be obtained fromInvitrogen, catalog # PHG9114; TIMP-1 may be obtained from R&D Systems,catalog #970-TM-010; and TIMP-2 may be obtained from R&D Systems,catalog #971-TM-010. VEGF, Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2are added to a carrier such as normal saline, PBS, lactated Ringer'ssolution, cell culture media, water or other suitable aqueous solutionknown to skilled artisans.

Example 4 Generation of Sustained-Release Compositions

Sustained-release compositions of ACCS, including pooled ACCS, or PCS,are produced by combining ACCS, including pooled ACCS, or PCScompositions with any of the sustained-release formulation technologiesdescribed herein (see Detailed Description) or otherwise familiar toskilled artisans.

Example 5 Use of ACCS to Prevent Onset of Periodontal Disease in anAnimal Model

Model: ACCS was tested in a rabbit model of P. gingivalis-inducedperiodontitis. The model utilized ligature+P. gingivalis applicationover a six week period which induces a considerable amount of gingivalinflammation and bone loss associated with periodontal disease as theconfirmation of the disease model. ACCS was applied prior to P.gingivalis application and then 3 times a week over the next 6 sixweeks.

Results: Topical application of ACCS resulted in a significant level ofprotection from inflammatory changes in soft tissue and bone lossinduced by ligature+P. gingivalis. These data indicate that ACCS caneither prevent or ameliorate the onset of periodontal disease. Placebo(unconditioned medium) application did not have any protective action asindicated by a significant amount of soft tissue inflammation and boneloss which was similar to that seen in the untreated group.

Example 6 Use of ACCS to Stop Progression of or Reverse PeriodontalDisease in an Animal Model

Model: ACCS is tested in a rabbit model of P. gingivalis-inducedperiodontitis. The model utilizes ligature+P. gingivalis applicationover a six week period which induces a considerable amount of gingivalinflammation and bone loss associated with periodontal disease as theconfirmation of the disease model. ACCS is applied 6 weeks afterligature+P. gingivalis application and then 3 times a week over anadditional 6 six weeks.

Example 7 Use of AMP Cells in an Animal Model of Periodontal Disease

Model: AMP cells are tested in a rabbit model of P. gingivalis-inducedperiodontitis. The model utilizes ligature+P. gingivalis applicationover a six week period which induces a considerable amount of gingivalinflammation and bone loss associated with periodontal disease as theconfirmation of the disease model. It is expected that AMP cells willhave the same positive effect (prevent or reverse or stop progression ofdisease) as ACCS because AMP cells secrete the active factors present inACCS.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Throughout the specification various publications have been referred to.It is intended that each publication be incorporated by reference in itsentirety into this specification.

What is claimed is,:
 1. A method for preventing, reversing, amelioratingor treating a dental disease, disorder or injury in a patient in needthereof, the method comprising the step of administering to the patienta therapeutically effective amount of one or more compositions selectedfrom the group consisting of extraembryonic cytokine-secreting (ECS)cells, conditioned medium derived therefrom, cell lysate derivedtherefrom, cell products derived therefrom, and physiologic cytokinesolution (PCS).
 2. The method of claim 1 wherein the dental disease isselected from the group consisting of gingivitis and periodontitis. 3.The method of claim 1 wherein the ECS cells are Amnion-derivedMultipotent Progenitor (AMP) cells.
 4. The method of claim 1 wherein theconditioned medium is Amnion-derived Cellular Cytokine Solution (ACCS)or pooled ACCS.
 5. The method of claim 1 wherein the PCS is formulatedfor sustained-release.
 6. The method of claim 4 wherein the ACCS orpooled ACCS is formulated for sustained-release.
 7. The method of claim1 wherein the ECS cells, conditioned medium derived therefrom, celllysate derived therefrom, or cell products derived therefrom areadministered in combination with other agents or treatment modalities.8. The method of claim 7 wherein the other agents are active agents. 9.The method of claim 8 wherein the active agents are selected from thegroup consisting of growth factors, cytokines, inhibitors,immunosuppressive agents, steroids, chemokines, antibodies, antibiotics,antifungals, antivirals, mitomycin C, and other cell types.
 10. Themethod of claim 7 wherein the other treatment modalities are selectedfrom the group consisting of non-surgical and surgical treatmentmodalities.
 11. The method of claim 10 wherein the non-surgicaltreatment modalities are selected from the group consisting ofprofessional dental cleaning, scaling and root planning
 12. The methodof claim 10 wherein the surgical treatment modalities are selected fromthe group consisting of flap surgery/pocket reduction surgery, bonegrafts, tissue engineering, soft tissue grafts, guided tissueregeneration, and bone surgery.